Kuo Chien-Wen, Gök Caglar, Fulton Hannah, Dickson-Murray Eleanor, Adu Samuel, Gallen Emily K, Mary Sheon, Robertson Alan D, Jordan Fiona, Dunning Emma, Mullen William, Smith Godfrey L, Fuller William
School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
School of Natural Sciences, College of Health and Science, University of Lincoln, Lincoln, UK.
Nat Commun. 2025 Feb 7;16(1):1445. doi: 10.1038/s41467-025-56716-x.
The complexity of the cellular proteome is massively expanded by a repertoire of chemically distinct reversible post-translational modifications (PTMs) that control protein localisation, interactions, and function. The temporal and spatial control of these PTMs is central to organism physiology, and mis-regulation of PTMs is a hallmark of many diseases. Here we present an approach to manipulate PTMs on target proteins using nanobodies fused to enzymes that control these PTMs. Anti-GFP nanobodies fused to thioesterases (which depalmitoylate protein cysteines) depalmitoylate GFP tagged substrates. A chemogenetic approach to enhance nanobody affinity for its target enables temporal control of target depalmitoylation. Using a thioesterase fused to a nanobody directed against the Ca(v)1.2 beta subunit we reduce palmitoylation of the Ca(v)1.2 alpha subunit, modifying the channel's voltage dependence and arrhythmia susceptibility in stem cell derived cardiac myocytes. We conclude that nanobody enzyme chimeras represent an approach to specifically manipulate PTMs, with applications in both the laboratory and the clinic.
细胞蛋白质组的复杂性通过一系列化学性质不同的可逆翻译后修饰（PTM）得到极大扩展，这些修饰控制着蛋白质的定位、相互作用和功能。这些PTM的时空控制对于生物体生理功能至关重要，而PTM的调控异常是许多疾病的一个标志。在此，我们提出一种利用与控制这些PTM的酶融合的纳米抗体来操纵靶蛋白上PTM的方法。与硫酯酶（使蛋白质半胱氨酸去棕榈酰化）融合的抗绿色荧光蛋白（GFP）纳米抗体可使标记有GFP的底物去棕榈酰化。一种增强纳米抗体对其靶标亲和力的化学遗传学方法能够实现对靶标去棕榈酰化的时间控制。使用与针对Ca（v）1.2β亚基的纳米抗体融合的硫酯酶，我们降低了Ca（v）1.2α亚基的棕榈酰化水平，改变了干细胞来源的心肌细胞中通道的电压依赖性和心律失常易感性。我们得出结论，纳米抗体 - 酶嵌合体代表了一种特异性操纵PTM的方法，在实验室和临床中均有应用。
